The Research Of Urine-derived Stem Cells Combined With Polycaprolactone/Gelatin Nanofibrous Scaffold On Skin Tissue Injury

Objective1.To study the human urine-derived stem cells (UDSCs) biologicalcharacteristics, and provide new and rich source of seed cells for regenerativemedicine;2. Construction of tissue-engineered skin with UDSCs and PCL/gelatin(PCL/GT) electrospun nanofiber membrane, then evaluate its impact on skin woundhealing capabilities.Methods1. To observe biological characteristics of UDSCs.1.1Isolation of UDSCs from the healthy donor flesh urine using self-madeconditional medium (LMM101medium)；1.2To identify surface antigen phenotype of UDSCs: CD29, CD90, CD73,CD34, CD146, HLA-DR by flow cytometry；1.3To identify multilineage potential of UDSCs：induced UDSCs in separateosteogenic, adipogenic and chondrogenic medium, followed by identifying with OilRed O, Alizarin Red and toluidine blue；2. The preparation of PCL/GT electrospun nanofiber membrane: mixingelectrospun of polycaprolactone (poly (ε-caprolactone), PCL) and gelatin (Gelatin,GT). Characterization of this material using Fourier transform infrared spectroscopy,contact angle, Young’s modulus and degradation rate.3. To observe biocompatibility of PCL/GT electrospun nanofiber membrane:After seeding UDSCs on PCL/GT membrane, cell proliferation was tested by CCK-8method; the cell survival rate on PCL/GT scaffold surface was detected by thefluorescent microscope after live/dead cell staining;then UDSCs combined withPCL/GT scaffold was observed using a scanning electron microscope(SEM).4. Construction of UDSCs combined with PCL/GT scaffold and transplantationin vivo:4.1After lentivirus with GFP infected4to6generations of UDSCs, UDSCsseeded on PCL/GT scaffold to transplant in vivo;4.2The full-thickness skin defects models and transplant:make the full-thicknessskin defect models(area:2cm×2cm)on the back of New Zealand White rabbits. All rabbit models were randomly grouped, then covered with UDSCs-PCL/GT scaffoldand PCL/GT scaffold, respectively. The control group was covered with Saline gauze.At14day, to observe the wound healing and calculate the wound closure rate. Tookthe wound and surrounding wounds tissue to make frozen sections, then observed thechanges in structure of skin tissue with HE staining and Masson’s trichrome staining,respectively. Lastly, detecting the changes in epithelial tissue and angiogenesis aswell as the survival of the (GFP+)UDSCs in vivo by Immunofluorescence.5. The mechanism of UDSCs combined with PCL/GT scaffold for repair skindamage: observed effect of UDSCs conditioned medium on proliferation,migrationand tube formation ability of human umbilical vein endothelial cells(HUVECs) byCCK-8test,Real Time Cellular Analysis(RTCA),scratch test and tube formationtest,to further Analyze the mechanism of UDSCs combined with PCL/GT scaffold forrepair skin damage.Results1. UDSCs successful could be isolated from fresh human urine. the cellsgrown to adherence after3-8days,colony-like after8-12days, and uniform offibroblast-like after2-3weeks subculture. Flow cytometry analysis showed thatUDSCs expressed CD146, CD29, CD90and CD73, but failed to express CD34andHLA-DR. It showed that UDSCs accords with characteristic of mesenchymal stemcells. In vitro induced UDSCs in separate osteogenic, adipogenic and chondrogenicmedium.After being cultured in osteogenic induction medium21days, Alizarin Redstaining displayed positive. After adipogenic induction medium14days, Oil Red Ostaining displayed positive. After chondrogenic induction medium28days,cellsgrown to cell pellet and toluidine blue staining displayed positive.The results showedUDSCs have the potential of differentiate into osteogenic, adipogenic andchondrogenic lineage.2. Successful preparation of PCL/GT electrospun nanofiber membrane.Hydrogen gelatin nitrogen (NH) deformation vibration absorption peak was observedat1500-1550cm-1and1600-1650cm-1by analysis of Fourier transform infrared,PCL or gelatin carbon-oxygen double bond characteristic (C=O) absorption peakwas at1700-1760cm-1, indicating successful preparation PCL/GT hybrid nanofibermembrane. The contact angle test results showed that the contact angle of PCL fibrous scaffolds for waterwas109°±0.4°,while PCL/GT fiber contact angledecreased to0°at25℃. The results showed that hydrophilic of PCL fibers could beenhanced by blending into the GT, and this was beneficial for improving thebiocompatibility of PCL fiber membrane material. Young’s modulus results showedPCL/GT fiber mechanical properties was very close to human skin tissue.Furthermore, in simulated body fluid, the weight loss of PCL/GT membrane reached90%in six weeks, while the weight loss of PCL membrane was less than5%,indicating degradation microenvironment of gelatin could promote the degradation ofPCL in composite film.3. SEM and live/dead cell staining results showed that UDSCs could attach andgrow on the PCL/GT scaffold，and the cells spread well and migrate deep inside thePCL/GT scaffold. Moreover, CCK-8assay indicated that UDSCs seeded on PCL/GTscaffold show good proliferation efficiency.It suggested PCL/GT scaffold have agood biocompatibility.4. UDSCs-PCL/GT significantly promoted skin wound healing. At14days, thewound closure rate in the UDSCs-PCL/GT groups was much higherthan that in thecontrol (p<0.05) and PCL-GT(p<0.05)；To compare with control and PCL-GT group,HE and Masson staining results showed UDSCs-PCL/GT groups wound havecomplete skin structure, more epithelial tissue, larger area of new collagenfiber(p<0.01)and intact skin attachments that new dense hair follicles at subcutaneous.Immunoflurescence staining of cells showed the epidermal area of UDSCs-PCL/GTgroup was much larger than PCL/GT group (p <0.01), there was a significantdifference (p <0.01) compared with control group. At7and14days, compared withthe PCL/GT group and the control group, the microvessel density ofUDSCs-PCL/GT group was significant higher (p <0.01). Analysis of fluorescencemicroscope revealed that (GFP+) UDSCs of wound tissue widely expressed in7days,but basically invisible in14day. It was speculated that UDSCs could promoteangiogenesis through paracrine mechanism of stem cells further to enhance woundhealing.5. UDSCs conditioned medium could promote proliferation,migration and tubeformation ability of HUVECs.CCK-8test result showed UDSCs conditioned medium significant enhance HUVECs proliferation efficiency;RTCA and scratch test showedUDSCs conditioned medium affect HUVECs and promote it migration; Results oftube formation test displayed UDSCs conditioned medium increase tube of capillariesnumber.These results suggested the paracrine factors of UDSCs be connected withangiogenesis ability of HUVECs.Conclusion1. Our research successfully has established the technology ofisolation and UDSCs accords with characteristic of mesenchymal stem cells. UDSCshas the abundant source, non-injury and autologous advantages. It was expected tobecome a new source of seed cells for regenerative medicine;2. PCL/GT nanofiberscaffold prepared in this study has good mechanical property and biocompatibility,and it could satisfy the demands of skin tissue engineered scaffold;3.UDSCscombined with PCL/GT scaffold enhanced skin wound healing,and it’s mechanismthrough paracrine mechanism of stem cells promote angiogenesis in injured tissuebeneficial to wound healing.